This disclosure relates to nucleic acid molecules that can be used to effect a loss of function of the von Hippel-Lindau (VHL) allele in somatic and/or germ cells of a mammal.
Von Hippel-Lindau (VHL) disease is an inherited multi-system disorder characterized by abnormal growth of blood vessels in certain parts of the body, including certain areas of the brain, retina, spinal cord, and adrenal glands. Lesions in the retina can cause retinal detachment and eventual blindness. Cataracts and glaucoma can occur as well. Angiomas in the brain or spinal cord may press on nerve or brain tissue and cause symptoms such as headaches, problems with balance, or weakness of arms and legs. Neurologic symptoms, including seizures and mental retardation, also may be present. However, symptoms vary greatly and depend on the size and location of the growths.
VHL also predisposes individuals to develop tumors of the kidney, adrenal gland, retina, and central nervous system. An autosomal dominant disorder, VHL is the most common form of inherited clear cell renal carcinoma. The multisystem character of the illness, combined with the fact that multiple tumors may form in each target organ, produces considerable morbidity and mortality; the life expectancy of affected individuals is only 49 years (McKusnick, V. A., Mendelian Inheritance in Man (1983) Johns Hopkins University Press. Baltimore and London, p. 534-535).
VHL disease is caused by germline mutations in the VHL tumor suppressor gene on chromosome 3p25. There is a need for a murine model for VHL disease to provide a tool for understanding how inactivation of the VHL gene can lead to the initiation and progression of tumor development in multiple organs. Such an animal model will also serve as a model system for testing tumor regression agents and gene therapies to improve the prognosis and quality of life of VHL patients. Recently, a mouse has been produced with a homozygous VHL gene deletion (Gnarra et al., PNAS 94:9102-9107, 1997). Because that mouse had an embryonic lethal phenotype, it did not provide a model for VHL disease.
Disclosed herein is a construct that includes at least three recombining sites, the first and second of which flank a selectable marker, and the second and third of which flank an exon of a VHLgene.
In one embodiment, the construct is used to generate a conditional deletion of an exon of a VHL gene. The method for producing the conditional deletion includes targeting the construct via homologous recombination to the endogenous VHL gene in embryonic stem cells, and using these stem cells to produce a conditional VHL targeted animal. In the presence of the appropriate recombinase, the recombining site-flanked portion of the VHL gene is excised. When the recombinase gene is placed under the control of a tissue-specific promoter, expression of the recombinase is directed in a tissue-specific manner and the recombining site-flanked portion of the VHL gene is deleted only in certain tissues.
The foregoing and other features and advantages will become more apparent from the following detailed description of several embodiments, which proceeds with reference to the accompanying figures.